Bovine antigen glycoprotein, related antibody, and use in detection of pregnancy in cattle

ABSTRACT

A bovine pregnancy antigen, determined to be a glycoprotein, has been isolated and purified. If is diagnostic for the presence of pregnancy in cattle when detected by the use of antibodies to the antigen.

This invention relates to a bovine pregnancy antigen, to a process forisolating it, to its use to create a diagnostic assay for the detectionand supervision of pregnancy in cattle, and to an antibody related tothe bovine pregnancy antigen.

BACKGROUND OF THE INVENTION

Detection of pregnancy in cattle, especially dairy cattle, is of greateconomic importance. Production of the hormone moiety required for milkproduction depends on a routine regimen of pregnancy for each cow.Toward this goal, and for a number of other reasons, 70% of the dairycows in the U.S. are artificially inseminated. Conception can only occurif insemination occurs at the proper stage of the estrous cycle of thecow. Since this receptive phase of the estrous cycle lasts only arelatively short time, an unsuccessfully bred cow can not bereinseminated until estrus (heat) occurs in the following cycle (21 dayslater). An important objective of the dairy industry, therefore, is todetect those animals in which a successful pregnancy was not establishedso that they may be rebred at the next cycle. If detection occurs beforethe first cycle is complete, the cow can be reinseminated in the nextheat, about 21 days after the first attempt. If detection is notpossible in that time, reinsemination must wait until the second cycle,42 days after the initial insemination or the third cycle at 63 days.The economic impact of having non-pregnant cattle for these extendedperiods of time can be substantial.

Presently, the majority of pregnancy detection in cattle is performed byveterinarians. The method used is a rectal palpation where the vetphysically feels for the presence of a developing fetus by "slipping" ormanipulating the membranes of the placenta. This technique only becomeseffective, however, in the period between 30 and 42 days of pregnancy.Depending on where in that time interval detection is made, at leastone, and perhaps two cycles could be missed. There are other problemswith the rectal palpation method: first, the vet has to be present atthe farm; and second, while the data are inconclusive, it has beenreported that there is a perception on the part of some farmers that themechanical manipulation required with rectal palpation might increasethe risk of spontaneous abortion.

Another method of pregnancy detection is the assay of progesterone inmilk. Progesterone is a steroid which is necessary for normaldevelopment and maintenance of pregnancy. Serum titers of progesteronerise throughout early stages of pregnancy, and increased levels of thehormone are found in milk. A radioimmunoassay (RIA) can detect theincrease in milk progesterone. This technique has a number ofshortcomings. By definition, an RIA requires the use of radioactivematerial. Inherent in this requirement are several disadvantages,including the need for sophisticated laboratory equipment, preventingboth the farmer and most veterinarians from performing the test. Inaddition, the level of progesterone varies both between normalnon-pregnant cows and within individual cows throughout the estrouscycle. Therefore, baseline readings for each cow are necessary, and atleast two determinations are required to confirm pregnancy. Furthermore,this method is reported to be unreliable earlier than 20 days afterinsemination.

It is clear that a more simple, rapid and accurate means of detectingpregnancy in cattle would be of great value.

The present inventors have reported, J. Animal Science, 57, Supp. 1, 320(1983), the initial isolation and partial characterization of proteinspecific to the chorionic membrane of bovine embryos. This protein wasreported to be a high molecular weight compound which, at one point inthe isolation process, appeared to stain as a glycoprotein. However,this protein had not been fully structurally characterized orsufficiently purified to be utilized for practical purposes.Furthermore, it had not been reported or suggested that such a proteinwould be found in the body components of the mother or would bediagnostic for pregnancy, or provide the basis for a pregnancy assay.

It has not been hitherto known that a bovine pregnancy antigen can beisolated from the embryo or body components of pregnant cattle, or thatsuch an antigen could serve as the basis for a diagnostic pregnancyassay.

OBJECTS OF THE INVENTION

An object of the present invention is a process for isolating a bovinepregnancy antigen by fractionation from the embryo or body components ofpregnant cattle. Another object of the present invention is the bovinepregnancy antigen itself. Yet another object of the present invention isan antibody specific to the bovine pregnancy antigen.

A further object of the present invention is a diagnostic assay for thedetection and supervision of a bovine pregnancy, which contains as anessential diagnostic component the bovine pregnancy antigen and/orantibody specific for the bovine pregnancy antigen.

GENERAL DESCRIPTION OF IMMUNOCHEMICAL REACTION TECHNIQUE

In general the production of antibodies to a substance and the use ofthe antibodies in assays for the substance is a practice well known tothe art. The antibodies may be polyclonal or monoclonal, although thespecificity of the monoclonal antibodies is generally advantageous andthey are usually utilized when available. The present invention relatesto the finding of a previously heretofor unrecognized bovine pregnancyantigen, antibodies specific thereto and the use of the latter indetecting pregnancy in cattle. The diagnostic assay of the invention maybe used for either the qualitative or quantitative detection of thebovine pregnancy antigen.

SUMMARY OF THE INVENTION

The present invention relates to (1) a novel bovine pregnancy antigenindicative of pregnancy in cattle, (2) methods for the isolation,purification and characterization of the antigen, (3) the use of theantigen to provide assays useful for the early detection of pregnancy incattle, (4) the preparation of polyclonal and monoclonal antibodies tothe antigen, (5) the use of the antibodies of the invention to provideassays useful to detect the antigen, and (6) pregnancy detection kitscomprising one or more components described above.

The bovine pregnancy antigen for detecting and determining pregnancy incattle is a glycoprotein obtained from a pregnant bovine animal and ischaracterized by

(a) binding nicotinamide-adenine dinucleotide,

(b) having active portions with a molecular weight of about 158,000 to263,000 daltons,

(c) containing at least the carbohydrates N-Ac-glucosamine, galactose,and L-fucose and either D-mannose or D-glucose or both,

(d) having an isoelectric point range of 4.5 to 5.5, and

(e) giving a blue stain with Coomassie Blue, and

(f) antibodies to which hemoglobin, albumin, bovine luteinizing hormone,IgG, fibrinogen, fetuin, and alpha fetoprotein are not cross-reactive.

DETAILED DESCRIPTION OF THE INVENTION PREPARATION OF BOVINE PREGNANCYANTIGEN

For preparing the bovine pregnancy antigen, bovine chorionic fluid wasconcentrated by precipitation in 50% aqueous ammonium sulfate solution.The precipitate was resuspended and desalted either by exhaustivedialysis in buffer (0.01M Tris, 0.15M NaCl, pH 7.5), or by passagethrough a Bio-gel P-2 column. An aliquot of the desalted sample wastested with saturated barium sulfate to assure that removal of ammoniumsulfate was complete. Protein concentration was determined by the methodof Lowry et al., J. Biol. Chem., 193, 265 (1951) or by the Bradfordmicroassay (BioRad Laboratories, Richmond, CA). Although the followingdescription relates to isolating the bovine pregnancy antigen frombovine chorionic fluid or the chorionic membrane, the antigen can bepurified also from the maternal blood of a pregnant cow.

A sample of the ammonium sulfate-treated material was mixed with anequal volume of 2M perchloric acid. The soluble fraction, deduced to bemostly glycoproteins by virtue of its solubility in perchloric acid, wasthen dialysed against phosphate buffered saline before determiningprotein concentration and immunoreactivity.

The ammonium sulfate chorionic fluid preparation was applied to aSephadex G-200 column (Pharmacia Fine Chemicals, Uppsala, Sweden).Samples were collected in 2 ml fractions and a chromatograph recordedabsorbance at 280 nanometers, as the concentrated sample was eluted fromthe column.

Alternatively, a sample of the ammonium sulfate chorionic fluidpreparation was applied to a column of Sephacryl S-300 (Pharmacia FineChemicals Inc.). This acrylamide cross-linked dextran matrix facilitatesthe resolution of large molecular weight components (limits:10,000-1.5×10⁶ daltons). Samples were again collected in 2 ml fractionsand absorbance of the fractions at 280 nanometers was recorded.

The desired bovine pregnancy antigen, a glycoprotein, was found byimmunoreactivity to be located primarily in a column fraction 43. Thisfraction 43 represented a fairly broad peak, and the maximum molecularweight was estimated to be 500,000 daltons. A more complete view of thisfraction was obtained in a 3%-27% gradient SDS-polyacrylamide gel and10% SDS polyacrylamide gel. These gels demonstrated that under reducingconditions this immunoreactive peak was composed of 3 to 4 bands,ranging in molecular weight from 220,000 to 600,000 daltons. Subsequentanalysis showed active portions of the antigen at molecular weights ofabout 158,000 to 263,000 daltons; see Example 15 infra.

Electrophoretic techniques were employed to more fully understand thenature of the bovine pregnancy antigen. Native and denaturingSDS-polyacrylamide gels with T values of 8%, 10%, 12%, and 3%-27%gradient were run and the gels were stained with either Coomassie Blueor silver stain by methods known to the art.

Electrophoretic patterns of fraction 43 revealed 3 to 4 bands. Theseincluded a doublet around 220,000, a large, ill-defined band around400,000 and one or more bands which contain material with a molecularweight too large to enter the 10% gel. These latter bands appeared inthe uppermost regions of the 3%-27% gel. The doublet at 220,000 closelyresembled the pattern of fibrinectin when treated under similarconditions. The large component at 400,000 resembled a glycoprotein inits staining behavior with Coomassie Blue. This component has also beenfound to bind to nicotinamide-adenine dinucleotide (NAD), which ischaracteristic of a glycoprotein.

It has been determined that the portion of the chorionic fluid whichreacts with antibodies must correspond to the 400,000 dalton componentof fraction 43. The bovine pregnancy antigen is a glycoprotein andpreliminary isoelectric focusing studies indicate the protein is acidic.

The bovine pregnancy antigen can be readily detected in pooled bloodsera from superovulated cows 13 days pregnant.

PREPARATION OF POLYCLONAL ANTIBODIES

Polyclonal antibodies to the bovine pregnancy antigen were prepared asfollows: Rabbits were immunized against concentrated chorionic fluid(CCF) using standard methods as described in more detail in theExamples. After immunization was complete, rabbit serum which wasdetermined to react with the marker bovine pregnancy antigen wascollected on a regular basis and stored for later use.

PREPARATION OF MONOCLONAL ANTIBODIES

Monoclonal antibodies to the marker bovine pregnancy antigen wereprepared as follows:

Step. 1. Preparation of a Monoclonal Antibody

The monoclonal antibodies useful in the present invention are obtainedby a process similar to that discussed by Milstein and Kohler andreported in Nature, 256: 495-497, 1975. The details of this process arewell known in the art and will not be repeated here. However, what isinvolved is injecting a mouse (or other suitable animal) withimmunogenic material. In the present invention, that material is apartially or completely purified glycoprotein. The immunized animals aresacrificed and cells from their spleens are fused, e.g., with mousemyeloma cells.

The result is a hybrid cell, known as a "hybridoma", that is capable ofreproducing in vitro. The population of hybridomas is then screened forimmunoglobulin production. Any of the several known methods forscreening for immunoglobulins such as those described in U.S. Pat. No.4,016,043 may be used. The enzyme-linked immunosorbent assay methoddescribed in U.S. Pat. No 4,016,043, using commercially availablereagents from several sources (such as Cappel Company) was found to beconveniently applicable. The immunoglobulins present in the cell culturefluids were further examined for their ability to react with theglycoprotein (bovine pregnancy antigen) used for immunization. This canbe accomplished by modifications of the above-mentioned enzymeimmunoassay.

Step 2. Identify Cell Clone

A specific clone producing the identified antibody, e.g., antibodies tothe bovine pregnancy antigen, can be propagated by maintaining it in asuitable medium and at a suitable temperature. A representative exampleis the use of Dulbecco's minimum essential medium (DME) in the presenceof 10% carbon dioxide and 10% fetal calf serum at 37° C.

Step 3. Antibody Isolation

The proteins, i.e., antibodies used in the assay, are obtained bytreating the tissue culture fluids from the spleen cell-myeloma cellfusion described above with 50% ammonium sulfate. This treatmentresulted in the precipitation of the desired antibodies. The precipitateis optionally (and preferably) resuspended in a buffered saline solutionfor further use.

Step 4. Antibody Immobilization

The antibodies obtained to the bovine pregnancy antigen are identified,propagated, isolated as above and then used, e.g., by immobilization orin solution. For example, antibodies may be adsorbed onto various waterinsoluble matrices such as microtiter plates, Dextran beads, nylon web,glass, cellulose, polyacrylamide, charcoal, urethane, ceramic, ormixtures thereof.

The antibody may be bound to the matrix by various methods known to theart, for example chemically, i.e., by the formation of ionic or covalentbonds or physically, i.e., by adsorption, entrapment in an insolublematrix, and the like.

The bound antibody can then be provided in a kit wherein body fluidsfrom female bovine animals would be added and activity of the antibodywith the fluids could be measured.

DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 are plots of optical density against serum dilutions(dilution curves) for determining pregnancy in cattle using thetechniques of the present invention. They are discussed in Examples 10and 14, infra.

The following examples are representative of the invention.

EXAMPLE 1 Collection of Embryonic Tissues and Fluids

The reproductive tracts of 355 pregnant cattle were obtained from alocal abattoir immediately after slaughter of the animals and returnedto the laboratory as quickly as possible. To reduce the possibility ofsample cross-contamination, tissues were collected in the followingorder. First the uterus was carefully sliced open and thecotyledon/caruncle interdigitations gently pulled apart to allow theintact placenta to fall away free. Using a 21 gauge needle and a 15 mlsyringe, a 12 ml sample of chorionic fluid was withdrawn and the fluidstored at -20° C. until needed. Similarly, other fluids were sampled andother tissues preserved.

EXAMPLE 2 Collection of Adult Bovine Samples

Bovine blood serum was collected by jugular venous puncture, allowed toclot overnight at 4° C., and then centrifuged at 1000×g for 15 min.Serum was drawn off with a disposable pipette, pooled (at least 6animals per sample), and stored at -20° C.

EXAMPLE 3 Tissue Homogenates

Pooled samples (at least 10 specimens) of chorion, from Day 45 ofgestation were weighed, minced, and suspended in 20% weight/volume coldphosphate-buffered saline (PBS). Each preparation was homogenized for 2min. (30 sec. bursts) in a Brinkman Polytron Tissue Homogenizer(Brinkman Instruments, Inc., Westbury, N.Y.). An ice pack was maintainedaround each tissue container during homogenization.

EXAMPLE 4 Embryonic Fluids

Chorionic fluid samples were pooled and then concentrated toapproximately 1 mg protein/ml by the addition of Lyphogel (GelmanInstrument Company, Ann Arbor, MI.) to each dilute solution. Lyphogel isa dry, selectively absorbent polyacrylamide material which absorbs aprecise multiple of its own weight in water, salt, and other smallmolecules (rejection limit 20,000 MW) from aqueous solution. The pH andosmolarity were unaffected. After the desired amount of waterabsorption, the Lyphogel was removed from each sample by suctionfiltration.

Alternatively, small chorionic fluid samples were concentrated using anAmicon B15 clinical sample concentrator (Amicon Corp., Lexington, MA.)This disposable multiple ultrafilter concentrates macromolecularconstituents of dilute samples at a rejection limit of 15,000 MW.

Pooled, concentrated chorionic fluid samples from day 65-75 of gestationwere used for isolation of the bovine pregnancy antigen. Samples werefirst pooled and then concentrated 25-fold for chromatography.

EXAMPLE 5 Preparation of Antisera

Two mature New Zealand White rabbits (female) were immunized againsteach of the Day 45 of gestation tissue or fluid samples described inExamples 3 and 4 above. Equal volumes of tissue homogenate orconcentrated extraembryonic fluid were emulsified with Freund's adjuvant(Difco Laboratories Inc., Detroit, MI). Each rabbit was injectedintradermally with 1.0 ml of the emulsion at multiple sites over thescapular region. The initial injection employed Freund's completeadjuvant, composed of Arlacel A (mannide mono-oleate), Bayol F (paraffinoil), and Mycobacterium butyricum. All other injections includedincomplete adjuvant which did not contain the bacteria. The secondinjection was administered one week later and three booster injectionswere given at two week intervals.

Normal serum was obtained from each rabbit prior to injection. A pool ofnormal rabbit control serum consisted of blood from at least threeanimals. Rabbit sera were collected three days prior to the thirdimmunization and at weekly intervals thereafter. Rabbits were bled fromthe marginal ear vein. The blood was allowed to clot overnight at 4° C.,and then centrifuged for 15 min. at 1000×g. Serum was removed with adisposable pipette and stored at -20° C. until used.

EXAMPLE 6 Ammonium Sulfate Fractionation

Salting out with ammonium sulfate was performed (in a 4° C. room) at 33,50, 66, 80, and 100% saturation on fractions of pooled, concentratedchorionic fluid from Days 65-76 of gestation (see Example 4).

Cold ammonium sulfate was added in a dropwise manner with continuousmixing. Samples were stirred for 2 min. and then centrifuged at 4° C.for 10 min. at 12,000×g. The supernatant fluid was removed and theprecipitate redissolved in 0.005M PBS to the original sample volume. Allfractions were examined for absorbancy at 278 nm on a Beckman Model 25double-beam spectrophotometer (Beckman Instruments Inc., Fullerton, CA).Ouchterlony analysis determined which fraction reacted most stronglywith the rabbit antisera against chorionic fluid proteins.

EXAMPLE 7 Sephadex G-200 Chromatography

Proteins in bovine chorionic fluid from Days 65-75 of pregnancy wereseparated by column chromatography using Sephadex gel. Sephadex swellsin aqueous solutions giving a porous gel. Only molecules below a certainsize (exclusion limit) can enter the pores, while larger molecules mustpass through the column in the liquid phase outside the gel particles.These large molecules elute first. Smaller molecules penetrate the poresto varying degrees, dependent on their shape and size. Elution throughSephadex columns, therefore, occurs in order of decreasing molecularweights. The gel and column were prepared as described by Okonkwo(1981):

1. Seven grams of Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala,Sweden) were suspended in 0.005M PBS pH 7.4. The dry gel was allowed toswell in excess buffer with intermittent stirring and decantation for 24hours at 4° C.

2. A 25×65 cm column (LKB 2137, Bromma, Sweden) with two flow adaptorswas used.

3. The column was filled with buffer to a height of 5-10 cm. The swollengel, which was suspended in a volume of buffer approximately twice theexpected bed volume, was carefully poured into the reservoir and airbubbles were removed. Flow rate was adjusted to 15 ml per hour asrecommended for Sephadex G-200. The bed was stabilized by eluting withapproximately two bed volumes of eluant.

4. Standards (Pharmacia) were used for calibration of the column. Theproteins standards used were

    ______________________________________                                        Protein Std.                                                                              Mol. Wt        Source                                             ______________________________________                                        Aldolase    158,000        Rabbit Muscle                                      Ovalbumin   45,000         Egg White                                          Cytochrome C                                                                              27,270         Horse Heart                                        ______________________________________                                    

5. The column was loaded with a 2 ml sample of concentrated chorionicfluid from Days 65-75 of gestation, containing about 20 mg of protein,and was eluted with 250 ml of buffer. Five ml fractions were collectedat a rate of 15 ml/hr with a fraction collector. All fractions wereexamined for absorbancy at 278 nm.

EXAMPLE 8 Monoclonal Antibody Production

1. Fusion

a. Mice (Balb/c) were immunized i.p. with the rechromatographed 50%ammonium sulfate precipitate fraction of chorionic fluid (see Example 7)at weekly intervals. Three days after their final injection, the spleenswere aseptically removed from the immunized animals and placed inculture medium (Dulbecco's modified Eagle's medium with Hepes buffer (20mm), (Gibco Laboratories, Grand Island, N.Y.) with double antibiotics(200 mM L-glutamine and 50 mg/ml Gentamycin). Serum was also collectedat this time and stored at -20° C. for future use.

b. Spleens were washed in sequential petri dish baths (2×) of culturemedium and then placed in a final dish containing 10 ml of the mediumfor pulp removal. Clear, empty spleen cases were then discarded and thepulp and medium transferred to 15 ml conical bottom sterile plastic testtubes (Corning Glassworks, Corning, N.Y.).

c. Tubes were centifuged at 120×g for 3 min., the supernatant fluidremoved, and the pellet resuspended in 10 ml of fresh culture medium.Viable cells were counted by Trypan Blue exclusion. Spleen cells werediluted to 1×10⁸ cells and distributed into 16×125 mm plastic roundbottom tubes (Corning Glassworks, Corning, N.Y.).

d. NS-1 plasmacytoma cells in log growth phase at 90% or greaterviability were added to the tubes containing the spleen cells at aconcentration of 2.5×10⁷ NS-1 cells, and the tubes centrifuged at 120×gfor 3 min.

e. The supernatant fluid was removed from all tubes by suction. Thetubes were then tapped to break up the pellets and 1 ml of polyethyleneglycol (PEG) solution was added. This marked the start of PEG exposuretiming. Tubes were shaken gently to resuspend all cells in the PEG andthen were recentrifuged at 120×g for 5 min. Tubes were next removed tothe laminar flow hood (Labgard, NuAire, Inc., Plymouth, MN) and allowedto incubate for about 3 min. longer (Total PEG exposure equals 8 min.).

f. PEG was quickly removed by suction and 10 ml of the culture mediumadded to each tube without disturbing the pellet. Tubes wererecentrifuged at 120×g for 3 min. The supernatant fluid was againremoved by suction.

g. Tubes were tapped gently to break up the pellets and 10 ml of culturemedium with antibiotics and serum (500 ml Dulbecco's modified Eagle'smedium with Hepes buffer (Gibco), 50 ml heat-inactivated fetal calfserum (Gibco), 5 ml L-glutamine, and 0.5 ml gentamycin slowly added.While the tubes were allowed to sit, an additional 10 ml of this mediumfor each tube was added to a 150 cm² flask. The contents of all tubeswere then poured into the flask and the cells were allowed to recoverovernight at 37° C.

h. The following morning, cell viability was again determined and thecells redistributed at a concentration of 10⁶ /ml in HAT selectionmedium. This cell suspension was then pipetted (0.3 ml/well) into theinner 60 wells of a 96-well culture dish (Costar, Cambridge, MA). Theouter wells were filled with sterile water and the plates were sealedand placed in a 37° C. incubator (Forma Scientific 3916, Mallinckrodt,Inc., Marietta, OH) in large plastic bags.

i. Three days later, fresh HAT selection medium was added. Henceforthcultures were observed daily and the medium changed as required. Hybridsappeared 10-14 days after fusion and were expanded into larger culturedishes as needed.

2. Feeder Plates

Cells in culture do not survive well when their numbers are reducedbelow some critical level. Feeder plates provide environmentalconditions condusive to hybridoma growth from single cell dilutions.Feeder spleen cells are not immortal and die off in about two weeks.

a. Spleens were aseptically removed from non-immunized animals, washed,the pulp removed, cells centrifuged, resuspended, and viabilitydetermined as described above. Cell density was adjusted to 10⁶ cells/mland the suspensions were placed in a 150 cm² flask overnight at 37° C.

b. The unattached cells were gently resuspended and removed from theflask the following morning, centrifuged and resuspended at 5×10⁶ viablecells/ml.

c. This suspension was then rapidly pipetted (0.1 ml/well) into theinner 60 wells of a 96-well plate. The outside wells were again filledwith sterile water. These plates were then ready for either expansion orsubcloning use.

EXAMPLE 9

1. Expansion

To reduce the possibility of cross-contamination between wells withrepeated feedings, and to encourage colony growth, cells were moved tonew and/or larger welled plates as needed. At that time supernatantfluids can be readily harvested for testing.

a. Cells in the fusion plate wells were resuspended using a steriletransfer pipette and half of the suspension was removed to a 15 ml roundbottom tube. Six ml of HAT selection medium were added and the cellsredistributed to the inner 60 wells of a feeder plate.

b. Cells were fed fresh culture medium every second day and observeduntil hybrid colonies were 60-80% confluent. Supernatant fluid from eachwell was then tested for immunoglobulin production by ELISA techniquesas described in Example 10, infra.

c. The following day, supernatant fluid and cells were collected andpooled from all positive wells. Suspensions were centrifuged (5 min. at120×g), the supernatant fluid was removed and saved, and the cellsresuspended in fresh medium with antibiotics and either expanded andsubcloned, or frozen as safety stocks.

2. Subcloning

Each original fusion plate has the potential to produce hundreds ofhybridoma cells capable of secreting an antibody of interest. To achievemonoclonal antibody production, these cells must eventually be platedone to a well and allowed to grow. These pure clones are the desired endproduct of cell fusion.

a. Using cells from the expansion plate, a 1 ml suspension containing10⁶ cells in fresh culture medium in a plastic 15 ml round bottom tube(Corning Glassworks) was made up.

b. Nine ml of this medium were added and the tube inverted several timesto mix (now 10⁵ cells/ml).

c. Next, 0.1 ml was removed and added to 9.9 ml of the same medium in anew tube and mixed (10³ cells/ml).

d. Finally, 0.2 ml was removed and added to 30 ml of the medium in a newtube (5-7 cells/ml), mixed and distributed to feeder plates at 0.1ml/well. Final plating density about 0.6 cells/well.

e. Plates were refed in 2-3 days and observed daily until they reached60-80% confluency. Supernatant fluids were again tested forimmunoglobulin production.

EXAMPLE 10 Enzyme-Linked Immunosorbant Assay (ELISA)

ELISA utilizes enzyme-linked (second) antibodies for which visualizationcan be provided by a measurable color change. Use of a supported antigenincreases the ease of handling, while environmental risks, such asradiation exposure, are eliminated. ELISA methodologies are excellentfor the rapid, large scale screening required when working withhybridomas.

a. Costar 1/2 area microtiter plates were coated overnight at 4° C. with50 ul/well of the same bovine pregnancy antigen preparation used toimmunize the mice diluted in carbonate buffer (1.59 g Na₂ Co₃, 2.93 gNaHCO₃ and 0.2 g NaN₃ per liter distilled water, pH 9.6).

b. Before use, plates were washed thrice with PBS-Tween and flicked dry.

c. Antibody solution (50 ul) was added to each well and allowed toincubate at room temperature for 1 hr.

d. Plates were again washed thrice and flicked dry.

e. Next, 50 ul of peroxidase-conjugated Protein A which normally bondsto IgG (Kirkgaard and Perry Laboratories, Inc., Gaithersburg, MA),diluted 1:5000 in PBS-Tween (8.0 g NaCl, 0.2 g KH₂ PO₄, 2.9 g Na₂ HPO₄,12H₂ O, 0.2 g KCl, 0.5 ml Tween 20, per liter distilled water, pH 7.4),was added to each well and allowed to incubate 1 hour at roomtemperature.

f. Again plates were washed thrice with PBS-Tween and flicked dry.

g. Substrate (20 ml Citrate buffer, 10 mg o-phenylenediamine, 10 μl H₂O₂ immediately before use) was added (50 ul/well) and color allowed todevelop for 15-30 min.

h. Plates were read on Dynatech MR580 Microelisa Reader (DynatechLaboratories, Inc., Alexandria, VA).

Results are portrayed as dilution curves as shown in FIGS. 1 and 2, andexplained in Example 14, infra.

EXAMPLE 11

Serum samples were obtained prior to artificial insemination of eachcow, and again 12 to 15 days post-insemination. Pregnancy was determinedby rectal palpation after all samples had been collected. The results ofanalysis of the samples are shown in Table I. Several monoclonalantibodies were tested for each serum sample from each cow. Thedesignations D14B001 A2, D14B001 A4, D14B002 A3, D15B003 A3, and D15B003A4 are all monoclonal antibodies raised against the bovine pregnancyantigen.

                  TABLE I                                                         ______________________________________                                        Serum                  % Inhibition by                                        Sample                 Post Insemination                                      Number      Antibody   Sample                                                 ______________________________________                                        1           Polyclonal 45                                                                 Antiserum                                                         1           D14B001 A2 21                                                     1           D14B001 A4 39                                                     1           D15B003 A3 27                                                     1           D15B003 A4 16                                                                            x = 29.6, s = 12.1                                     2           D14B001 A2 53                                                     2           D14B001 A4 31                                                     2           D14B002 A3 22                                                     2           D15B003 A3 46                                                     2           D15B003 A4 17                                                                            x = 35.7, s = 18.7                                     3           Polyclonal 48                                                                 Antiserum                                                         3           D14B001 A2 52                                                     3           D14B001 A4 42                                                     3           D14B002 A3 28                                                     3           D15B003 A3 32                                                     3           D15B003 A4 57                                                                            x = 43.1, s = 11.4                                     4           Polyclonal 16                                                                 Antiserum                                                         5           Antiserum  12                                                                 polyclonal                                                        ______________________________________                                    

These results show that the cows that provided samples 1, 2, and 3 werepregnant. Sample 1 was 12 days pregnant, samples 2 and 3 were 15 dayspregnant, and Samples 4 and 5 were provided by non-pregnant cows. Theantibody was added with the serum samples to the wells which were coatedwith the bovine pregnancy antigen preparation. After incubation,peroxidase-labeled protein A was added. Following this reaction, colorchanging substrate was added and color change observed. These resultsdemonstrate that pregnant cows can be distinguished from non-pregnantcows by the method shown.

EXAMPLE 12

A 500 ml column was filled with Sephacryl S-300 and the column waspacked by running Tris buffer (0.01M trishydroxyethylamine, 0.15M sodiumchloride, pH 7.5) through at 0.667 ml/min. The column was calibratedusing standard proteins.

A 5 ml sample of concentrated chorionic fluid was obtained byprecipitation of chorionic fluid with 50% ammonium sulfate, separationof the insoluble fraction, and washing and redissolving with water. Thesample was then added to the column and eluted with Tris buffer.Fractions of 2.1 ml were collected and the flow rate was 0.24 ml/minute.A series of peaks were detected by optical density measurement at 280nanometers. The molecular weight is estimated by comparison with thestandards. Peaks were observed at greater than 600,000 (void volume),about 400,000, about 150,000, about 65,000, about 31,000 and about18,000 daltons.

EXAMPLE 13

The various fractions of the column run in Example 12, especially thefraction of about 400,000 daltons, were allowed to react with rabbitserum containing polyclonal antibodies to concentrated chorionic fluidcontaining the marker glycoprotein (see Example 5). A precipitant bandwas found with its apex at the 400,000 dalton peak. This resultindicates that this protein is the marker protein for pregnancy incattle.

EXAMPLE 14

Partially purified concentrated chorionic fluid (as obtained in Example13 and described hereinabove as fraction 43) was diluted 1:100 incarbonate buffer (pH 9.6) to be fixed into microtiter plate wells.

Rabbit antiserum was diluted from 1:10 to 1:780,000.

Serum was obtained from a cow prior to artificial insemination(non-pregnant) and also about 15 days after insemination (pregnant). Theserum samples were assayed using the method of Example 10.

It was found that the serum obtained from the pregnant cow blockedbinding markedly more than the serum obtained prior to artificialinsemination over the entire range of dilutions. The results are showngraphically in FIG. 1.

EXAMPLE 15

Using the general method of Example 12, but taking smaller fractions (1ml) of the eluate, a set of samples was obtained from 500 ml ofchorionic fluid clarified by centrifugation.

The set of samples contained 5 major peaks. When tested for the bovinepregnancy antigen in a counterimmunoelectrophoresis assay, all of theantigenic activity was contained in the second major peak. This majorpeak included the fraction designated 43 in earler separations. Theentire group of fractions corresponding to the second major peak waspooled and designated Fraction 2.

Samples of Fraction 2 were diluted with equal volumes of a buffercontaining 2-mercaptoethanol. The mixture was boiled for 5 minutes, thenapplied to a 5 to 15% linear gradient polyacrylamide gel, andelectrophoresed at 30 milliamps for 2 to 3 hours. The gels were theneither fixed and stained for protein (to assess molecular weight) orplaced in tris-glycine-methanol buffer and transferred byelectrophoresis to nitrocellulose paper (transblots). The transblotscould be stained for protein detection or reacted with variousantibodies.

Although the molecular weights as determined by this method are moreprecise, i.e. peaks at 158,500 daltons (major), 208,900 daltons(secondary) and 263,000 daltons (tertiary), they may possibly representpartial degradation of the antigen glycoprotein. Each of these peaksretains significant antigenic activity and thus represent majorconstituents or the only major constituent of the antigen.

EXAMPLE 16

A sample of Fraction 2 of Example 15 was evaluated by isoelectricfocusing on both agarose and polyacrylamide gels.

Using a 3.5 to 9.5 pH gradient on polyacrylamide gel, bands were foundat 4.7, 5.3, 5.45 to 5.8, and 6.6 to 6.75.

Using a 3 to 10 pH gradient on agarose gel, bands were found at 4.5, 5.3(a doublet), and 5.5. Therefore, the band or bands attributable to theglycoprotein antigen are in the range 4.5 to 5.5.

The reference pH values are determined using a surface electrode;glycoprotein bands are located on the fixed gel stained with CoomassieBlue dye.

EXAMPLE 17

Lectins are a group of plant proteins that specifically bind to sugarsor their derivatives. Since the bovine pregnancy antigen is aglycoprotein, a purification scheme based on differential binding toimmobilized lectins was possible.

Two types of survey experiments were performed, batch-wise and columnfractionation of the active chorionic antigen fraction. All experimentswere performed in 10 mM Tris, 150 mM NaCl, and 0.002% NaN₃ buffer. Inthe batch experiments 100 μl of washed resin (immobilized lectin) wererocked with 150 μl of the test solution for 135 min at ambienttemperature. The supernatants were saved for antigen analysis. Thesefractions contained unbound protein. The resin was washed 4× with thebuffer, then rocked for 1 hr. with the corresponding sugar. Thesupernatant from this fraction was also saved for analysis, comprisingthose proteins which were specifically bound to lectin.

The column fractionation was similar to the batch experiment. A smallcolumn of resin was prepared in buffer. Three ml of test solution wereloaded onto the column followed by a buffer wash. Elution with thespecific sugar in buffer followed by 1% SDS (sodium dodecylsulfate, adenaturing detergent) in buffer was then carried out. These fractionswere analyzed for antigen activity.

The results of the column experiments reproduce the batch results andgave enough material for SDS-PAGE analysis. Separate ConA columns wereeluted with either α-methylmannoside or α-methylglucoside and yieldedidentical results. No antigen passed through the column; sugar elutedantigen as did SDS.

In the T. purpureas column experiment, antigen eluted in theloading/washing, sugar, and SDS fractions. Identical behavior was seenwith the WGA column.

Conclusion: The bovine pregnancy antigen contains the following sugars:N-Ac-glucosamine, galactose, L-fucose, and either D-mannose or D-glucoseor both.

    ______________________________________                                        Lectin               Sugar                                                    ______________________________________                                        Concanavalin A (ConA)                                                                              α-methylmannoside                                                       α-methylglucoside                                  Lentil               α-methylmannoside                                                       α-methylglucoside                                  T. purpureas         L-fucose                                                 Ricinis communis II (M.sub.r = 120K)                                                               galactose                                                (RCA)                                                                         Wheat germ           N--Ac--glucosamine                                       (WGA)                                                                         ______________________________________                                    

What is claimed is:
 1. An isolated, substantially pure bovine pregnancyantigen for detecting and determining pregnancy in cattle consisting ofa glycoprotein obtained from a pregnant bovine animal which(a) bindsnicotinamide-adenine dinucleotide, (b) has immunoreactive portions witha molecular weight of about 158,000 to 263,000, (c) contains at leastthe carbohydrates N-Ac-glucosamine, galactose, and L-fucose, and eitherD-mannose or D-glucose or both, (d) has an isoelectric point range of4.5 to 5.5, (e) gives a blue stain with Coomassie Blue, and (f) is notcross-reactive when antibodies directed to said glycoprotein arecontacted with hemoglobin, albumin, bovine luteinizing hormone, IgG,fibrinogen, fetuin, or alpha fetal protein.
 2. A method for detectingthe presence of a bovine pregnancy antigen in the blood serum, urine,milk, saliva, or tissue extract of a female bovine which comprises,contacting a sample of the blood serum, urine, milk or tissue extract tobe tested with antiserum to the bovine pregnancy antigen of claim 1 andexamining the sample for evidence of immunoreactivity.
 3. A monoclonalantibody produced by a hybridoma formed by fusion of cells from a mousemyeloma line and spleen cells from a mouse previously immunized with thebovine pregnancy antigen of claim 1, with which said antibody reacts. 4.A test kit, to be used for the detection and determination of pregnancyin cattle comprising(a) the monoclonal antibody of claim 3, (b)peroxidase-labeled protein A, and (c) color changing substrate.
 5. Amethod for the early detection of pregnancy in bovine animals comprisingcontacting a serum sample of a female bovine animal with the componentsof a test kit of claim 4 wherein a color change positively indicates thepregnancy of said female bovine animal.
 6. A method for the earlydetection of pregnancy in bovine animals comprising contacting a serumsample of a female bovine animal with the bovine pregnancy antigen ofclaim 1 wherein the subsequent presence of antibody-antigen complexespositively indicate the pregnancy of said female bovine animal.
 7. Themethod of claim 6 wherein bovine pregnancy is detected within one estruscycle.
 8. The method of claim 6 wherein bovine pregnancy is detected12-15 days after insemination.
 9. The method of claim 6 wherein bovinepregnancy is detected 13 days after insemination.
 10. A process forisolating a bovine pregnancy antigen comprising(a) combining chorion orchorionic fluid from pregnant cows with an inorganic physiologicallycompatible salt whereby a first precipitate is formed, (b) resuspendingand desalting said first precipitate to form a concentrated fluidpreparation, (c) filtering said concentrated fluid preparation on ananion-exchanger whereby a bovine pregnancy antigen is adsorbed on saidanion-exchanger, (d) eluting said antigen from said anion-exchanger toform a crude glycoprotein of about 400,000 molecular weight, and (e)recovering said antigen of about 400,000 molecular weight.